Method and device to control and condition the behavior of the varroa destructor mite in a beehive, or in other environment where bees reproduce

ABSTRACT

Method and device to control and condition the behavior of the Varroa destructor mite in a beehive, or in another environment where bees reproduce. The method comprises a step of applying, in the beehive or in the aforesaid environment, a chemical composition including at least one of the following substances: (+)-2-hydroxyhexanoic acid, (−)-2-hydroxyhexanoic acid, (+)-3-hydroxyhexanoic acid, (−)-3-hydroxyhexanoic acid, or a mixture thereof.

FIELD OF THE INVENTION

The present invention concerns a method and device to control andcondition the behavior of the Varroa destructor mite in a beehive, or inartificial means associated with the reproduction of bees such as cells,observation plates, or any other environment suitable for thereproduction of bees. To be more exact, the method provides to use achemical composition comprising (±)-2-hydroxyhexanoic acid,(±)-3-hydroxyhexanoic acid, or a mixture thereof, which is able toattract the Varroa destructor mite and can thus be used to condition thebehavior of the Varroa destructor mite in the beehive in order toprevent it from reproducing.

BACKGROUND OF THE INVENTION

It is known that the Varroa destructor mite is a parasite on thedomestic bee Apis mellifera that causes enormous damage to the beecolonies, so much so that, without a suitable protection treatment, thefamilies of bees succumb in a short time to the action of this parasite.

Numerous acaricides are known in the struggle against this mite,including for example fluvalinate, flumethrin, acrinathrin, amitraz,coumaphos and other similar molecules.

Such acaricides, however, have the disadvantage that, since they have nospecific action against the Varroa destructor mite, they can causecollateral damage to the bees; moreover, they have lost theireffectiveness in many geographical areas, since many Varroa destructorpopulations have developed a resistance to one or more acaricides andcan therefore continue to reproduce on the bees.

Another shortcoming of known acaricides is that they leave unwantedresidues in the products of the bees such as the honey and the wax.

The accumulation of residues in the wax is worrying in that it is alsoused as a component in pharmaceutical and cosmetic creams and pomadesand hence it must not have any unwanted residue of acaricide.

For these reasons, numerous studies have been carried out on substancesknown as semio-chemicals (chemical signals), involved in the life cycleof the Varroa destructor mite, in order to use them to modify thebehavior thereof and to reduce the levels of infestation. Thesesubstances are present naturally in the beehive, they act in extremelylow concentrations and are often volatile, so that the problem ofresidues does not exist.

In particular, great attention has been paid to the parasite'sreproduction process, which occurs inside the cells containing thedeveloping bee larva. The Varroa destructor mite enters the cell a fewhours before opercolature and is induced, among other things, bychemical stimuli arriving from the cell itself. This behavior isexploited, in bee-keeping techniques, to capture part of the Varroadestructor mite on a honeycomb, called honeycomb trap, containing aclutch which, after opercolature, is removed from the nest and thendestroyed, thus reducing the infestation; this intervention, however,has limited effectiveness and hence does not solve the problem.

The chemical stimuli involved in the entrance of the mite into the cellhave been the object of numerous studies which have led to theidentification of generic substances, such as methyl- and ethyl-estersof fatty acids, such as methyl palminate, aliphatic alcohols, saturatedhydrocarbons with an odd number of carbon atoms, such as C₁₉-C₂₉, but inno case have the substances identified shown activity in the beehive,allowing to perfect devices to trap the mite.

Applicant has devised and embodied the present invention to overcomethese shortcomings of the state of the art and to obtain otheradvantages.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized essentially in themain claims, while the dependent claims describe other innovativecharacteristics of the invention.

Purpose of the invention is to perfect a method by means of which it ispossible to condition the behavior of the Varroa destructor mite in abeehive or in any other environment where bees reproduce, in order toprevent the clutch of bee larvae from being destroyed by the mite.

Another purpose of the present invention is to achieve a device thatcontains a semio-chemical substance active against the Varroa destructormite, and that at the same time is able to release this substance slowlyover time, in order to ensure a long-term protection of the beehive.

In accordance with these purposes, a method according to the presentinvention provides that a chemical composition comprising at least oneof the following substances: (+)-2-hydroxyhexanoic acid,(−)-2-hydroxyhexanoic acid, (+)-3-hydroxyhexanoic acid,(−)-3-hydroxyhexanoic acid, or a mixture thereof, is applied in adeterminate zone of a beehive or of means associated with thereproduction of bees, such as for example cells, observation plates, orany other environment suitable for the reproduction of bees, so as toattract the Varroa destructor mite therein.

It is known that these substances: (+)-2-hydroxyhexanoic acid and(−)-2-hydroxyhexanoic acid, and similarly (+)-3-hydroxyhexanoic acid and(−)-3-hydroxyhexanoic acid, are respectively optically active isomers.In the following description of the invention and of some preferentialforms of embodiment of the invention, the two optical isomers(+)-2-hydroxyhexanoic acid or (−)-2-hydroxyhexanoic acid are groupedtogether for the sake of simplicity in the single expression“(±)-2-hydroxyhexanoic acid”, this expression meaning that, according tothe invention, either one of the two individual isomers is used, or theracemic mixture of the two.

The expression “(±)-3-hydroxyhexanoic acid” will be used to indicate theuse of one of the two single optical isomers, (+)-3-hydroxyhexanoicacid, or (−)-3-hydroxyhexanoic acid, or the use of the racemic mixtureof the two.

To be more exact, (±)-2-hydroxyhexanoic acid and (±)-3-hydroxyhexanoicacid are attractive substances that attract the mite and are not toxicfor the bees because they are contained in the food supplied to the beelarvae by the nursing bees.

By introducing an attractive substance into a beehive, or into theaforesaid artificial means, it is possible according to the invention tocondition the behavior and the movement of the mite inside them, inorder to prevent it from entering into the cells where there are the beelarvae. Advantageously the method provides that the chemical compositionis first prepared by dissolving (±)-2-hydroxyhexanoic acid, or(±)-3-hydroxyhexanoic acid, or a mixture thereof, into a solvent,preferably water, or into other solvents such as esters of fatty acids,alcohols, or otherwise, able to facilitate the application thereof.

A preferential embodiment of the invention provides to prepare thechemical composition by means of one or more stabilizers able to protectin particular the hydroxyl group of (±)-2-hydroxyhexanoic acid, or of(±)-3-hydroxyhexanoic acid, and ensure that said acids, once applied inthe beehive or in said means associated with the reproduction of thebees, do not deteriorate over time, because of contact with air orlight.

In one embodiment of the invention, the chemical composition isdispersed, for example by means of a nebulizer, directly onto a portionof the clutch, so as to attract the Varroa destructor mite thereon.Subsequently, after opercolature, the portion of clutch is destroyed soas to completely eliminate the mite.

According to a variant, the chemical composition is applied directly indeterminate areas of the beehive, or of said artificial means, which donot contain any clutches, so as to attract the Varroa destructor mite toareas not suitable for it to reproduce.

According to a variant, the chemical composition is applied not directlyin said areas without clutches, but on predetermined small-size supportmeans or artificial substrates, such as a plate, a disk, a panel, ableto be arranged inside the beehive or inside said artificial means.

According to another variant, the method according to the presentinvention provides to saturate the area of the beehive by means of thechemical composition containing (±)-2-hydroxyhexanoic acid,(±)-3-hydroxyhexanoic acid, or a mixture thereof.

To be more exact, the method comprises a preparation step to prepare adevice provided with containing means to contain the aforesaid chemicalcomposition, and delivery means connected to the containing means andable to release (±)-2-hydroxyhexanoic acid, (±)-3-hydroxyhexanoic acid,or a mixture thereof, slowly over time. The device comprises for examplea capsule, a wrapper, a container, a boat or a sponge and is insertedinside the beehive or between the artificial means.

The slow and constant release of (±)-2-hydroxyhexanoic acid or of(±)-3-hydroxyhexanoic acid or of both allows to confuse the Varroadestructor mite, interfering with its ability to identify the clutchsuitable for reproduction.

According to another variant, the method according to the inventionprovides to introduce the composition containing (±)-2-hydroxyhexanoicacid, (±)-3-hydroxyhexanoic acid, or a mixture thereof, into thebeehives or into the aforesaid artificial means during a period when theclutch is absent, for example in winter, so as to stimulate the Varroadestructor mite to search for the clutch to infest and thus begin itsreproductive cycle during an unfavorable period, thus indirectly causingit to die.

DESCRIPTION OF THE DRAWINGS

These and other characteristics of the present invention will becomeapparent from the following description of some activity tests of theaforesaid chemical composition and of some preferential forms ofembodiment of the invention, given as a non-restrictive example. Withreference to the attached drawing, FIG. 1 shows an artificialobservation plate used to perform a biological activity test of(±)-2-hydroxyhexanoic acid and of (±)-3-hydroxyhexanoic acid. To be moreexact, the activity tests were performed for the racemic mixtures of(±)-2-hydroxyhexanoic acid and of (±)-3-hydroxyhexanoic acid.

Activity test of (±)-2-hydroxyhexanoic acid of (±)-3-hydroxyhexanoicacid in artificial observation plates

To perfect the method according to the present invention an activitytest was made of (±)-2-hydroxyhexanoic acid and of (±)-3-hydroxyhexanoicacid in the laboratory, in artificial observation plates.(±)-2-hydroxyhexanoic acid was tested in a beehive too, as will bedescribed hereafter.

By means of this activity test it is proved that said acids arenaturally present in the food supplied to the bee larvae by the nursingbees and contained in the cells. To be more exact, said acids wereextracted in acetone to isolate them from the rest of the larva food andwere then tested according to the biological test described hereafter.

The activity of (±)-2-hydroxyhexanoic acid and of (±)-3-hydroxyhexanoicacid, dissolved in water, was tested in a plate 10 (FIG. 1). Each plate10 consists of a parallelepiped of sodium-calcium glass with a 70×70 mmsquare base, and a height of 12 mm, in which four cylindrical wells 12a, 12 b, are made, each with a diameter of 7 mm and a depth of 10 mm,similar in size to bee cells. The wells 12 a, 12 b are equidistant fromthe center. At the center of the plate 10 a Varroa destructor mite wasplaced, indicated by the number 11.

To delimit the space in which the mite 11 could move, the plates 10 wereclosed with a cover element 15 consisting of a glass disk joined to astainless steel spacer ring 17. Two opposite wells 12 a of each plate 10were treated with (±)-2-hydroxyhexanoic acid or with(±)-3-hydroxyhexanoic acid, dissolved in water; the other two wells 12b, on the contrary, were used as controls and were treated only with thesolvent. In each individual well 12 a, 12 b of the plate 10 a bee larva20 was then placed.

The biological activity test on (±)-2-hydroxyhexanoic acid provides afirst test during which 1 ng of (±)-2-hydroxyhexanoic acid is tested, asecond test during which 10 ng of (±)-2-hydroxyhexanoic acid are testedand a third test during which 100 ng are tested and a fourth test duringwhich 1000 ng are tested.

The biological activity test on (±)-3-hydroxyhexanoic acid provides afirst test during which 10 ng of (±)-3-hydroxyhexanoic acid are tested,a second test during which 100 ng of (±)-3-hydroxyhexanoic acid aretested, and a third test during which 1000 ng are tested.

At the beginning of each test the Varroa destructor mite 11, in thiscase a female adult, is located at the center of each plate 10 with athin brush; each plate 10 is then closed and transferred to athermostatic chamber. Subsequently, the position of the mite 11 wasmonitored every 5 minutes for a total of 30 minutes, to a total of 6observations.

20 plates were used for every test, with 4 repetitions, to a total of 80plates. During the tests the temperature and relative humidity were keptrespectively around 34° C. and 75%; the inside of the thermostaticchamber was kept dark.

The action of the substances tested on the Varroa destructor mite wasevaluated by calculating an average value for the treated wells 12 a andthe control wells 12 b, equal to the average number of times in whichthe Varroa destructor mite was observed inside one or the other in the 6observations made in the course of the 30 minutes of each test. Theaverage values thus obtained are representative, with a certainapproximation, of the time spent by the mite in the two types of well 12a, 12 b. The attractiveness of a substance thus determines higheraverage values for the treated wells 12 a with respect to those of thecontrol wells 12 b. It must be pointed out that, for every test, the sumof the average values of the treated wells 12 a and of the control wells12 b does not reach a total of 100% because a fraction, more or lessgreat, of the mites spends part of the time outside the wells 12 a, 12b.

(±)-2-hydroxyhexanoic acid was found to be active on and attractive tothe Varroa destructor mite at doses of 10 and 100 ng, since itdetermined a significantly greater infestation of the treated wells 12 awith respect to the control wells 12 b.

(±)-3-hydroxyhexanoic acid was found to be active on and attractive tothe Varroa destructor mite at the dose of 100 ng, since it determined asignificantly greater infestation of the treated wells 12 a with respectto the control wells 12 b.

Comparative Test

Other substances naturally present in the food supplied to bee larvae bythe nursing bees, identified by extracting and isolating them from thelarva food, were also tested by means of the activity test.

To be more exact, the activity test was done at least three times with100 ng of each of the following substances: hexanoic acid, octanoicacid, nonanoic acid, phenylacetic acid, benzoic acid, (Z)-3-hexanoicacid, (E)-3-hexanoic acid, 2-methylbutanoic acid, 3-methylbutanoic acid,3-methylpentanoic acid and 2-ethylhexanoic acid.

To give an example, we report the results of the comparative testobtained with (±)-2-hydroxyoctanoic acid which has a very similarstructure to (±)-2-hydroxyhexanoic acid. 100 ng of (±)-2-hydroxyoctanoicacid were tested with respect to the control and this acid was not foundto be active.

The results of the activity test of (±)-2-hydroxyhexanoic acid and of(±)-3-hydroxyhexanoic acid and of the comparative test are reported inTable 1; to be more exact, the activities of (+)-2-hydroxyhexanoic acidand of (±)-3-hydroxyhexanoic acid at different doses were compared witheach other and with (±)-2-hydroxyoctanoic acid. TABLE 1 Result of somelaboratory tests with substances identified in the larva food. AVERAGESIGNIFI- ACTIVITY CANCE ACTIVE PRINCIPLE TREATED CONTROL(±)-2-hydroxyhexanoic 22.3 23.5 — acid, 1 ng (±)-2-hydroxyhexanoic 31.817.8 P < 1% acid, 10 ng (±)-2-hydroxyhexanoic 34.7 21.1 P < 1% acid, 100ng (±)-2-hydroxyhexanoic 23.0 26.0 — acid, 1000 ng (±)-3-hydroxyhexanoic24.5 21.8 — acid, 10 ng (±)-3-hydroxyhexanoic 36.8 22.8 P < 1% acid, 100ng (±)-3-hydroxyhexanoic 23.3 22.5 — acid, 1000 ng (±)-2-hydroxyoctanoic29.5 29.5 — acid, 100 ng

Activity test of (±)-2-Hydroxyhexanoic acid in the beehive

(±)-2-hydroxyhexanoic acid was also tested in the beehive, by applying,in the cells with worker bee larvae near to opercolature, a solution of100 ng of (±)-2-hydroxyhexanoic acid dissolved in 1 μl of a suitablesolvent. The infestation of such cells was compared with that of thecontrol cells treated with the solvent alone: the cells used wereexamined 18 hours after treatment and the Varroa destructor mitespresent in the treated cells and in the control cells were counted.

The number of Varroa destructor mites per cell in the cells treated with(±)-2-hydroxyhexanoic acid was significantly greater than that found inthe non-treated cells.

This means that (±)-2-hydroxyhexanoic acid modifies and conditions thebehavior of the Varroa destructor mite in the beehive too. TABLE 2Result of the tests carried out in the beehive with(±)-2-hydroxyhexanoic acid N. OF VARROA DESTRUCTOR MITE N. OF CELLS PERCELL REPLICATION TREATED TREATED CONTROL 1 225 0.27 0.22 2 265 0.32 0.233 180 0.30 0.26 4 169 1.38 0.72 5 140 0.73 0.70 Average 0.60 0.43

DETAILED DESCRIPTION OF SOME PREFERENTIAL FORMS OF EMBODIMENT OF THEINVENTION

According to a first form of embodiment, the method according to thepresent invention provides a first step wherein a chemical compositionC1 is prepared with a concentration of about 0.100 g/l of(±)-2-hydroxyhexanoic acid, or of (±)-3-hydroxyhexanoic acid, or amixture thereof, in water.

In a second step, 1 μl of the composition C1 is injected into each cellof a group of cells of a beehive with worker bee larvae near toopercolature. In this way we obtain a group of cells, the so-calledtreated cells, which attract the Varroa destructor mites.

After opercolature, the group of treated cells is extracted from thebeehive and destroyed, so that the Varroa destructor mites are alsodestroyed with it.

According to another form of embodiment, the method according to thepresent invention provides a first step wherein a chemical compositionC1 is prepared with a concentration of about 0.100 g/l of(±)-2-hydroxyhexanoic acid or (±)-3-hydroxyhexanoic acid or a mixturethereof, dissolved in water.

In a second step, the composition C1 is nebulized onto a group of cellsin a beehive with worker bee larvae near to opercolture. In this way weobtain a group of cells, the so-called treated cells, which attract theVarroa destructor mites.

After opercolature, the group of treated cells is extracted from thebeehive and destroyed, so that the Varroa destructor mites are alsodestroyed with it.

According to another form of embodiment of the invention, the methodprovides to prepare a chemical composition C2 containing(±)-2-hydroxyhexanoic acid or (±)-3-hydroxyhexanoic acid or a mixturethereof, with an overall dose of about 10 mg, and other compoundsincluding stabilizers of a known type, which ensure the protection ofthe hydroxyl group and the conservation of the acids over time.

The chemical composition C2 is then inserted in a delivery device suchas, for example, a capsule of a known type, which is subsequentlypositioned inside the beehive in order to release (±)-2-hydroxyhexanoicacid and/or (±)-3-hydroxyhexanoic acid slowly and progressivelythroughout the environment.

Diffused (±)-2-hydroxyhexanoic acid and/or diffused(±)-3-hydroxyhexanoic acid confuse the Varroa destructor mite so as toprevent it from identifying the clutch of bee larvae, so that it can nolonger reproduce.

Therefore, the use of one of the following substances:(+)-2-hydroxyhexanoic acid, (−)-2-hydroxyhexanoic acid,(+)-3-hydroxyhexanoic acid, (−)-3-hydroxyhexanoic acid, or of a mixturethereof, is able to condition and control the behavior and the movementof the Varroa destructor mite in a beehive or in artificial meansassociated with the reproduction of bees.

It is clear, however, that modifications and/or additions of parts canbe made to the method as described heretofore without departing from thefield and scope of the present invention.

1. Method to control and condition the behavior of the Varroa destructormite in a beehive, or in artificial means associated with thereproduction of bees, comprising a step of applying, in said beehive, orin said artificial means, a chemical composition comprising a substanceselected from the group consisting of: (+)-2-hydroxyhexanoic acid,(−)-2-hydroxyhexanoic acid, (+)-3-hydroxyhexanoic acid,(−)-3-hydroxyhexanoic acid, of and a mixture thereof.
 2. Method as inclaim 1, wherein said chemical composition is dispersed on a portion ofa clutch in said beehive, or in said artificial means, so as to attractthereon the Varroa destructor mite.
 3. Method as in claim 2, whereinsaid portion of said clutch, after an opercolature step, is destroyed soas to eliminate the Varroa destructor mite.
 4. Method as in claim 1,wherein said chemical composition is applied directly in determinateareas of said beehive, or of said artificial means, where there is noclutch of bee larvae, so as to attract the Varroa destructor mite tozones not suitable for the mite to reproduce.
 5. Method as in claim 1,wherein said chemical composition is applied on substrates or artificialsupport means, which are then arranged in determinate areas of saidbeehive, or of said artificial means, where there is no clutch of beelarvae, so as to attract the Varroa destructor mite to zones notsuitable for the mite to reproduce.
 6. Method as in claim 1, whereinsaid chemical composition is applied in zones of said beehive, or ofsaid artificial means, without a clutch, so as to stimulate the Varroadestructor mite to begin its reproductive phase during an unfavorableperiod and thus indirectly causing the mite to die.
 7. Method as inclaim 1, comprising a saturation step of said beehive, or of saidartificial means, during which said chemical composition is dispersed inrandom manner so as to confuse the Varroa destructor mite, interferingwith ability of the mite to identify a clutch of bee larvae.
 8. Methodas in claim 1, comprising a first preparation step to prepare saidchemical composition wherein stabilizing compounds are added to one ormore of the following substances: (+)-2-hydroxyhexanoic acid,(−)-2-hydroxyhexanoic acid, (+)-3-hydroxyhexanoic acid,(−)-3-hydroxyhexanoic acid.
 9. Device for the application of a chemicalcomposition in a beehive, or in artificial means associated with thereproduction of bees, to condition the behavior of the Varroa destructormite, comprising containing means to contain a chemical compositioncomprising a substance selected from the group consisting of:(+)-2-hydroxyhexanoic acid, (−)-2-hydroxyhexanoic acid,(+)-3-hydroxyhexanoic acid, (−)-3-hydroxyhexanoic acid, and a mixturethereof, and delivery means connected to said containing means and ableto deliver one of said substances or the mixture thereof.
 10. Device asin claim 9, wherein said containing means and delivery means comprise acapsule, a container, a boat or a spongy element able to progressivelyrelease over time at least one of said substances or the mixturethereof.
 11. Device as in claim 9, wherein said containing means alsocontain stabilizing substances able to protect said(+)-2-hydroxyhexanoic acid, said (−)-2hydroxyhexanoic acid, said(+)-3-hydroxyhexanoic acid, or said (−)-3-hydroxyhexanoic acid, from thedegradation which can occur in contact with air, or due to exposure tolight.
 12. A method of use of a substance selected from the groupconsisting of: (+)-2-hydroxyhexanoic acid, (−)-2-hydroxyhexanoic acid,(+)-3- hydroxyhexanoic acid, (−)-3-hydroxyhexanoic acid, and a mixturethereof, in order to condition and control the behavior and the movementof the Varroa destructor mite in a beehive or in artificial meansassociated with the reproduction of bees comprising depositing thesubstance at a location to cause the mite to be attracted to thelocation.